There are several disorders associated with changes in the bone resorption rate, the most widely spread being osteoporosis, a disease common in postmenopausal women. Osteoporosis is caused by loss of bone mass resulting in a weak skeleton, which is susceptible to fractures. The treatment of fractures and other maladies caused by osteoporosis is expensive to society, not to mention the suffering of the patients. There is therefore a need for diagnosing persons suffering from osteoporosis in order to prevent severe consequences thereof. Osteoporosis has been successfully treated e.g. by estrogen replacement or biphosphonate treatment.
There are several instruments and methods for measuring bone density. However, the apparatuses and operations used are expensive, because they require much space and personnel and the measuring is slow and the patient has to be present. Since the age-class distribution of the population is becoming more unfavourable as far as osteoporosis is concerned, it is important to develop specific, rapid, simple and cheap in vitro methods for measuring the bone resorption rate.
Bone consists substantially of two major cell types, the bone forming osteoblasts and the bone resorbing osteoclasts. Normally these two cell types are in balance, the osteoclasts resorb the same amount of bone as the osteoblasts form. Osteoporosis is the result of an increased activity of the osteoclasts resulting in a disorder where the osteoclasts resorb more bone than the osteoblasts produce.
Tartrate-resistant acid phosphatase (TRAP, EC 3.1.3.2.) belongs to the type 5 class of acid phosphatases according to its electrophoretic mobility and is therefore also called type 5 acid phosphatase. At least seven acid phosphatases can be identified by acidic polyacrylamide gel electrophoresis. The most acidic of these is the band 5 acid phosphatase (Acp5), and it is the only one resistant to inhibition by tartrate, therefore the name TRAP. This enzyme is a basic glycoprotein that contains a spin-coupled iron unit at the active site of the molecule. The binuclear iron unit of TRAP contains two ferric ions, which make the protein purple, and it is also called purple acid phosphatase. This protein has a molecular weight of 32 kd. When the enzyme is reduced e.g. with .beta.-mercaptoethanol, one of the ferric ions is reduced and the enzyme becomes pink. TRAP can function as a protein tyrosine phosphatase in vitro and its amino acid sequence contains regions homologous to those of phosphoprotein phosphatases. However, its natural substrates are still unknown.
The tyrosine phosphorylation is an important regulation mechanism for the function and differentiation of many cells. TRAP is believed to play an important role in the regulation of the function of the osteoclasts. It is known that TRAP is capable of producing hydroxyl radicals, which are capable of reacting and destroying chemical compounds in nature. The osteoclasts produce free oxygen radicals during bone resorption and it is evident that they are significant in bone resorption. Their origin is unknown, but it seems that at least part of the hydroxyl radicals is formed by TRAP. Both cell types normally containing TRAP i.e. the osteoclasts and the alveolar macrophages take up chemical compounds and degrade them. This common feature strongly supports the assumption that TRAP takes part in bone resorption.
Hayman, A. R. et al. (Development 122:3151-3162, 1996) have investigated the role of TRAP by targeted disruption of the TRAP gene in mice. They found that mice lacking the gene had disrupted endochondral ossification and mild osteopetrosis. They concluded that TRAP is required for normal mineralization of cartilage in developing bones and that it also maintains integrity and turnover of the adult skeleton by a critical contribution to bone matrix resorption.
The only normal human cells that contain significant amounts of TRAP are the osteoclasts and activated macrophages. In certain pathological conditions TRAP can also be found in other cells. It has been found that TRAP is secreted from the osteoclasts into the blood circulation during bone resorption. The concentration of TRAP in serum has therefore been suggested as a marker of bone resorption, and it has been found that the concentration of TRAP in serum correlates with the bone resorption rate (Kraenzlin M. E. et al. Journal of Clinical Endocrinology and Metabolism 71(2):442-451, 1990; Cheung C. K. et al. Clin. Chem. 41(5):679-686, 1995; Chamberlain P. et al. Clin. Chem. 41(10):1495-1499, 1995 and Halleen J. et al. Journal of Bone and Mineral Research 11(10):1444-1452, 1996).
In 1978 Lam W. K. W. et al. (Clin. Chem. 24(7):1105-1108, 1978) reported a study of the biochemical properties of TRAP in the serum of adults and children. By an improved electrophoretic method they found two distinct TRAP bands, which they designated 5a and 5b. TRAP 5a had a lower pH optimum than TRAP 5b. Both enzyme forms TRAP 5a and TRAP 5b were found in human sera. The amount of TRAP 5a was found to be constant in adults and children, but the amount of TRAP 5b was elevated in children.
In another paper (Chen et al., Clin. Chem. 25, 719-722, 1979), the same group of investigators studied the significance of high acid phosphatase activity in the serum of normal children. They showed that serum TRAP activity, corresponding to band 5, was present in giant-cell tumors, and not in osteogenic sarcomas, suggesting that TRAP would be derived from osteoclasts.
W. K. W. Lam et al. (Clin. Biochem. 14, 177-181, 1981) isolated TRAP from the serum and spleen of patients affected by Gaucher's disease. They showed that Gaucher's disease serum contained band 5b, whereas Gaucher's disease spleen contained band 5a. Bands 5a and 5b had identical protein structure, and removal of carbohydrate from 5a by sialidase converted it to 5b, suggesting that the only structural difference between 5a and 5b would be the presence of sialic acid residues in 5a that are not present in 5b. The pH-optima of the two forms was different, being 5.0 for 5a, and 5.5-6.0 for 5b.
W. K. W. Lam and R. J. Desnick (Progress in Clinical and Biological Research 95, 267-278, 1982) further studied the band 5 forms in Gaucher's disease. They also summarized the electrophoretic profiles of acid phosphatases in normal and pathologic specimen. They showed that in normal serum, trace amounts of both 5a and 5b are present, and they did not observe elevation of band 5a in any pathologic serum. Instead, they found elevated amounts of 5b in osteoclastic bone tumors, in the serum of patients with malignancies metastasized to bone, and in the serum of patients with Gaucher's disease. Based on these results the authors concluded that the results might suggest that the serum acid phosphatase level, and in particular the 5b form, is primarily dependent on the physiologic activity of the osteoclasts. However, they stated that further proof to support the hypothesis was needed. An other important observation from the paper is the authors statement that the enzymes (bands 5a and 5b) are closely related and antigenically identical. According to this statement, it would not be possible to develop an immunoassay that would specifically measure either 5a or 5b. After the year 1982, the origin of serum TRAP 5a and 5b has not been studied further, and the suggested osteoclastic origin of 5b has not been verified.
TRAP activity can be determined spectrophotometrically e.g. by feeding p-nitrophenyl phosphate (pNPP) as a substrate in the presence of tartrate, which inhibits most other acid phosphatases. However, this method is not very specific, and therefore more specific methods i.e. immunoassays have been developed for the determination of TRAP.
Stepan J. J et al. (Biochem. Biophys. Res. Commun. 165:1027-1034, 1989) and Kraenzlin M. E. et al. (Journal of Clinical Endocrinology and Metabolism 71(2):442-451, 1990) have developed an immunoassay for TRAP using polyclonal antibodies raised against TRAP purified from the spleen of a patient with hairy cell leukemia. They suggest that their assay may be useful in detecting patients with disorders in mineral metabolism.
Cheung C. K. et al. (Clin. Chem. 41(5):679-686, 1995) disclose an immunoassay of TRAP using polyclonal antibodies prepared against TRAP isolated from cord plasma. They found that the concentration of TRAP was significantly higher in children and in postmenopausal women and discuss the possibility of using TRAP as a marker in assessing bone turnover.
Polyclonal antibodies have also been prepared against TRAP isolated and purified form human bone. Again it was proved that children and postmenopausal women had elevated concentrations of TRAP in their sera (Halleen J. et al. Journal of Bone and Mineral Research 11(10):1444-1452, 1996).
Chamberlain P. et al. (Clin. Chem. 41(10):1495-1499, 1995) disclose a two-step serum immunoassay of TRAP using a pair of monoclonal antibodies, which had been raised against recombinantly produced TRAP. The monoclonals recognized different epitopes of TRAP and enabled a sensitive assay thereof.
All of the immunoassays published so far measure the total content of TRAP in serum. However, the present invention is based on the finding that TRAP 5b is a much more specific biochemical marker for bone resorption than total TRAP. In this connection it should be pointed out that the isoforms of TRAP reported by Stepan et al. and Kraenzlin et al. supra should not be confused with those reported by Lam et al. supra, although both are designated 5a and 5b.
One object of the present invention is therefore to take advantage of TRAP 5b as a specific marker for bone resorption.
Another object of the present invention is to provide a specific and simple method of measuring the bone resorption rate and especially to provide a diagnostic method of diagnosing a disorder associated with a significant change in the bone resorption rate, such as osteoporosis or other metabolic bone diseases, bone lesions, or bone metastases.
One further object of the present invention is to provide a method which enables monitoring bone resorption in subjects suspectible to or suffering from these disorders and to follow up the effect of treatment of them.
Still another object of the present invention is to provide a test kit useful in said methods.